CN102335162A - Application of trichostatin A (TSA) to preparation of medicament for inhibiting activities of swine ovary granular cells - Google Patents

Abstract

The invention belongs to the field of medicaments and veterinary medicines and discloses application of trichostatin A (TSA) to preparation of a medicament for inhibiting activity of swine ovary granular cells. In the invention, the influence of the TSA to the activities of the swine ovary granular cells is systemically studied, and a result shows that the TSA has no influence to the growth of the swine ovary granular cells at low concentration (below 10ng/mL), with the increase of the concentration, the growth of the swine ovary granular cells is inhibited (P<0.05), and the inhibition effect is dose-dependent. After the granular cells are treated by using 200ng/mL TSA, the cell cycle is retardant within a period G0/G1, and the marked decrease of gene expression of Cyclin D2 and CDK4 (Cyclin-Dependent Kinase 4) is possible to be an important reason for the cycle retardance. Accordingly, the TSA can be applied to preparation of the medicament for inhibiting the activities of the swine ovary granular cells.

Description

Trichostatin A suppresses the application in the active medicine of pig ovary granular cell in preparation

Technical field

The invention belongs to medicine and field of veterinary, relate to Trichostatin A and suppress the application in the active medicine of pig ovary granular cell in preparation.

Technical background

Granular cell is crucial somatic cell in the ovary, and it is synthetic and secreting hormone and multiple somatomedin in the different phase of growing, and plays a role through autocrine/paracrine approach.The growth of granular cell and differentiation especially play crucial effects to the maturation of oocyte to the g and D of ovarian follicles.Follicular development begins to experience primary follicle, secondary follicle and the chamber follicle is arranged and finally becomes mature follicle from primordial follicle.

In each estrus cycle; All have a large amount of follicles to start growth in the Mammalian Ovary, but the overwhelming majority can not reach maturity, and degenerate gradually in each stage of development of follicle; Degeneration that takes place before this ovulation of follicle and the physiological phenomenon that finally is eliminated are called follicular atresia.Think that at present primordial follicle, primary follicle locking are mainly caused by the oocyte apoptosis.In the follicular development later stage, it possibly be that the granular cell apoptosis causes in oocyte self apoptosis or the follicle that the locking of chamber follicle and the preceding mature follicle of ovulation is arranged.Research at present thinks that primordial follicle, the primary follicle locking of most animals fetus mainly are to be caused by the oocyte apoptosis, and adult mainly to be the granular cell apoptosis cause.Big quantity research shows that apoptosis betides each stage of follicular development, and wherein the apoptosis of granular cell is the major reason that causes follicular atresia.If the granular cell generation apoptosis in development follicle more than 10% then shows this follicle or just in locking, and in a single day follicle gets into locking and just can not return on the normal growth track.

Trichostatin A (TSA) is that first comes to light and has the active natural product of inhibition of histone deacetylase.From the metabolite of Streptomyces hygroscopicus, be separated in 1976 by Tsuji the earliest, demonstrate the antifungal effect.Recent study finds that TSA has obvious suppression or lethal effect to kinds of tumor cells, as: stomach cancer cell, leukaemia, cervical cancer cell, breast cancer cell, colon cancer cell, endometrial carcinomas cell and prostate gland cancer cell etc.In addition, have multiple deacetylase inhibitor to get into clinical experimental study, therefore, in the near future, body contact deacetylase inhibitor (HDACi) can increase gradually, and amount also can be increasing.Do not see at present the relevant report of TSA as yet to the granular cell influence.

Cell proliferation is the basis of life entity breeding, also is the important way of adult individual damaged tissue repair cell.The propagation of body cell must be followed certain rules and carries out without any confusion, and receives tight control.The cell proliferation process comprises material preparation (preparing before the division) and two processes that connect each other of cell division; This material is prepared and cell division cyclic process continuously; Be called cell cycle; Claiming CDC again, is that phalangeal cell finishes to divide the overall process that end is experienced next time from once dividing.A complete cell cycle is divided into G1 phase, S phase, G2 phase and M phase successively.Wherein, the G1 phase is that the cell growth is synthesized incubation period with DNA for be accomplished to the intermittent time before the dna replication dna from mitosis.Get into the G1 after date, cell begins the required RNA of synthetically grown, protein, saccharide and lipid etc., and its volume also can increase gradually simultaneously.The S phase is dna replication dna period, and this phase content of DNA doubles.The G2 phase, this phase cell synthesized a large amount of protein, for mitosis is prepared in order to be accomplished to this intersegmental having a rest that mitosis begins from dna replication dna.The M phase is a cell division phase, and the cell division of this phase is two daughter cells, accomplishes one-step growth.

The change of cell proliferation situation is to realize through the regulating action of cyclin to cycle progression.The core protein of participating in cell cycle regulation in the eukaryote mainly is divided into 3 big types, is respectively cyclin (Cyclin), cell cycle protein dependent kinase (Cdk) and cell cycle protein dependent kinase inhibitive factor (CKI).Wherein, Cyclin periodically accumulates and decomposes the cell cycle process and plays pivotal role, and Cdk is the key link of Cycle Regulation.Cyclin is the positive regulating factor of Cdk, and CKI is the inhibitive factor of Cdk.

Summary of the invention

The objective of the invention is above-mentioned deficiency, provide Trichostatin A to suppress the application in the active medicine of pig ovary granular cell in preparation to prior art.

The object of the invention can be realized through following technical scheme:

Trichostatin A suppresses the application in the active medicine of pig ovary granular cell in preparation.

Preferred 100～the 200ng/mL of the concentration of Trichostatin A, further preferred 200ng/mL.

Beneficial effect:

Systematic study of the present invention Trichostatin A to the active influence of pig ovary granular cell; The result shows; TSA when low concentration (below the 10ng/mL) to not influence of the growth of pig ovary granular cell; But along with the increase of concentration, the growth of pig ovary granular cell is suppressed (P＜0.05), and this inhibitory action has dose dependent.When 10ng/mL and 200ng/mL, along with the prolongation in processing time, when 48h, slightly raise, when 72h, reduce again, but the OD550 value difference of each time point determining different not remarkable (P＞0.05).Growth does not have time dependence to the pig ovary granular cell to show TSA.Utilize the influence of indirect immunofluorescence research Antibiotic FR 901228 TSA to normal pig ovary granular cell acetylation level; And utilize fluorescent quantitative PCR technique mechanism to be done further research from molecular level, show that TSA influences the acetylation level of normal granules cell under higher concentration.After adopting flow cytometer detection variable concentrations Trichostatin A (TSA) to act on granular cell 24h, the situation of change in granular cell cycle, and utilize the fluorescence quantitative RT-RCR technology to study from molecular level cell cycle related gene.After showing that granular cell is handled with 200ng/mL TSA, cell cycle is arrested in the G0/G1 phase, and the remarkable reduction of Cyclin D2 and CDK4 gene expression possibly be the major reason of Cycle Arrest.Therefore Trichostatin A can suppress the application in the active medicine of pig ovary granular cell in preparation.

Description of drawings

Fig. 1 immunocytochemical technique method detects the influence that TSA handles back granular cell H3K9 acetylation level;

A: blank group; B:10ng/mL TSA processed group; C:200ng/mL TSA processed group.

Fig. 2 TSA handles the acetylizad relative quantity of back granular cell H3K9.

Fig. 3 immunocytochemical technique method detects the influence that TSA handles back granular cell H3K14 acetylation level;

A: blank group; B:10ng/mL TSA processed group; C:200ng/mL TSA processed group.

Fig. 4 TSA handles the acetylizad relative quantity of back granular cell H3K14.

Fig. 5 TSA handles the influence to granular cell acetylase related gene expression, and significant difference is significantly represented (P＜0.05) with * between each group.

Fig. 6 flow cytometry TSA handles the influence to the granular cell cycle;

A: blank group; B:10ng/mL TSA processed group; C:200ng/mL TSA processed group.

Fig. 7 TSA handles the influence that the granular cell cycle associated genes is expressed, and significant difference is significantly represented (P＜0.05) with * between each group.

The specific embodiment

Embodiment 1

Laboratory animal

Laboratory animal of the present invention is the general merchandise sow, is provided by ring pig slaughtering field, sky, Nanjing.

Reagent

DMEM/F12 cultivates dry powder: GIBCO company; Newborn hyclone: Lanzhou Min Hai company;

MTT cell proliferation and cytotoxicity detection kit: Kai Ji company; Antibiotic: Sigma company;

KCl, K ₂HPO ₄, NaCl, Na ₂HPO ₄Be analytical pure Deng chemical reagent: Chemical Reagent Co., Ltd., Sinopharm Group.

1.1 the collection of pig ovary and the selection of follicle

Pig ovary is gathered in ring slaughterhouse, sky, Nanjing, puts in 37 ℃ of normal saline, takes back laboratory in the 2h, gives a baby a bath on the third day after its birth time with the PBS of the interpolation penicillin of preheating and streptomycin (Sigma).Choose granular cell in the follicle as experiment material.

1.2 the acquisition of pig ovary granular cell

Granular cell obtains from follicle, uses the syringe that has No. 20 syringe needles from follicle, to draw follicular fluid (FD is 2-6mm), removes by filter oocyte with the filter screen (100 μ m) of aseptic steel.The centrifugal 10min of 800g, and wash twice with PBS, abandon supernatant.

1.3 it is foster that pig ovary granular cell former is commissioned to train

The cell precipitation of collecting is put into super-clean bench, light alcohol burner, open blower fan; Draw 1mL DMEM/F-12 to 1.5mL centrifuge tube with liquid-transfering gun, fully piping and druming is until being dispersed in existence; Cell counting, adjustment density to 10 ⁶Individual/mL; Be inoculated in the 25mL culture bottle, place 37 ℃, 5%CO ₂, cultivate in the 95% humidity incubator; Observation situation behind the 24h treats that cell grows to 80% and goes down to posterity when converging or be used for handling.

1.4 going down to posterity of pig ovary granular cell

From incubator, take out culture bottle, light rolling is blown and beaten, and pours out liquid after not adherent cell is all blown afloat; Add 2-3mL PBS, twice of repeated washing gone in light rolling; Add the trypsin solution about 1mL, jog makes it fully contact with adherent cell; After hatching 1-2min in the incubator, take out and examine under a microscope to most of cell levitating; Add the DMEM/F-12 that contains hyclone in right amount immediately and stop digestion, piping and druming is to suspending; Cell counting, adjustment density to 10 ⁶Individual/mL; Be inoculated in the new culture bottle marked respectively on former generation and the culture bottle that goes down to posterity; Place incubator to cultivate, observe growing state behind the 24h.

1.5 the pig ovary granular cell is frozen

(1) digests according to the method pair cell that goes down to posterity; (2) collect postdigestive cell in the centrifuge tube of 1.5mL; (3) the centrifugal 5min of 1000rpm abandons supernatant; (4) add an amount of cryopreserving liquid, piping and druming cell system suspends; (5) cell counting, the adjustment cell density is 10 ⁶Individual/mL; (6) be sub-packed in the frozen pipe, cere is sealed up for safekeeping; (7) annotated labelling, behind 4 ℃ ,-20 ℃ ,-80 ℃ placement 1h, 2h, 2h, put into liquid nitrogen container successively and preserve subsequent use.

1.6 the recovery of pig ovary granular cell

The frozen pipe that (1) will take out places 37 ℃ of water-baths rapidly, constantly shakes, and frozen cell suspension is melted as early as possible; (2) with pipet suspension is moved in the centrifuge tube; (3) DMEM/F-12 that adds 10 times of volumes blows and beats mixing; (4) the centrifugal 5min of 1000rpm abandons supernatant; (5) adding DMEM/F-12 piping and druming deposition system suspends; (6) cell counting, adjustment density to 10 ⁶Individual/mL; (7) be inoculated in the culture bottle marked; (8) place incubator to cultivate, observe growing state behind the 24h.

1.7MTT method is measured cell activity

Select the cell of exponential phase,, utilize trypan blue dyestuff discharge method to confirm cell viability, be mixed with single cell suspension with the DMEM/F-12 culture medium that contains 10%FBS with hematimeter pair cell counting; With 5 * 10 ⁴The density of individual/mL is inoculated in 96 orifice plates, every hole 100 μ L; Put 37 ℃, 5%CO2,95% humidity and cultivate 24h; Press Concentraton gradient then and add testing sample, each concentration is established 3 multiple holes, establishes blank and negative control simultaneously; Put 37 ℃, 5%CO ₂, after 95% humidity cultivates certain hour, adding concentration in every hole is the MTT 20 μ L of 5mg/mL, puts into incubator and continues to cultivate 4 hours; Stop cultivating, inhale and abandon culture medium in the hole, add dimethyl sulfoxide (DMSO) 150 μ L, put into incubator and hatch 20min, first a ceremonial jade-ladle, used in libation crystal is fully dissolved.Detect each hole light absorption value under the 550nm wavelength with ELIASA.With time is transverse axis, and the absorbance of each group of each time point is the longitudinal axis, draws cell growth curve.Test parallel three times for every group, independent triplicate adopts SPSS 16.0 statistical analysis softwares to carry out One-wayANOVA and analyzes, the LSD multiple comparisons, and data are all with average ± standard error (means ± SE) expression.The result sees table 1 and table 2.

Table 1. variable concentrations TSA handles 24h granular cell growing state

Table 2.TSA handles different time to the active influence of granular cell

The above results shows: dna methylase inhibitor inhibitors TSA (10ng/mL below) when low concentration does not influence the growth of pig ovary granular cell; But increase along with drug level; The growth of pig ovary granular cell is suppressed (P＜0.05); And this inhibitory action has dose dependent, and the result sees shown in the table 1.In addition, we have also detected the influence to the pig ovary granular cell at different time points TSA, and when 10ng/mL and 200ng/mL,, slightly all raise to some extent at 48h, but when 72h, reduce again along with the prolongation in processing time, but the OD of each time point determining ₅₅₀Value difference different not remarkable (P＞0.05) shows that TSA does not have time dependence to the inhibition of pig ovary granular cell growth, and is as shown in table 2.

Embodiment 2

Reagent

The glycogen of Trizol, no RNase: Invitrogen company; M-MLV Reverse Transcriptase:Promega company; The DEPC:Sigma packing; 2 * Taq PCR Master Mix: sky, Nanjing root Bioisystech Co., Ltd; Oligo dT, dNTP MixtureRNase inhibitor, SYBR Premix Ex TaqTM:Takara company; DNA marker:Takara company; Boric acid, agarose, Tris (Tris), bromophenol blue: the biological company limited of Nanjing great order; Chloroform, isopropyl alcohol, dehydrated alcohol are analytical pure: Chemical Reagent Co., Ltd., Sinopharm Group.

The solution preparation

(1) 10 * tbe buffer liquid storage liquid: Tris 108.99g, boric acid 55.62g, EDTA 29.23g are dissolved in the 1000mL distilled water, pH8.0-8.2, autoclaving, 4 ℃ or room temperature preservation.

(2) bromophenol blue solution: the 0.25g bromophenol blue is dissolved in the 100mL sterilization distilled water, 4 ℃ or room temperature preservation.

(3) 2 * sample-loading buffers: Tris-HCl 100mmol/L pH6.8, SDS 4.0% (W/V), bromophenol blue 0.2% (W/V), glycerol 20% (V/V), DTT 200mM.

(4) lysis buffer: 150mM NaCL, 50mM Tris, pH7.4,2mM EDTA, 0.5%Triton X-100,1mM NaVO31mM PMSF, 1mM NaF.

(5) TBST (Tris salt contains the Tween-20 buffer): Tris-Base 20mmol/L, NaCl 137mmol/L, Tween-200.1% (V/V), pH7.4.

(6) antibody confining liquid: the TBST buffer that contains 5%BSA or defatted milk powder.

2.1 the pig ovary granular cell is cultivated: with embodiment 1.

2.2 indirect immunofluorescence

(1) exponential phase pig ovary granular cell is inoculated in the culture dish that is placed with coverslip; When cell attachment grows to 70%-80%; TSA with 0ng/mL, 10ng/mL and 200ng/mL handles pig ovary granular cell 24h respectively, takes out coverslip, PBS flushing 3 times;

(2) be put in then among the PBS that contains 4% paraformaldehyde, room temperature is 15min fixedly, and PBS cleans 3 times, each 5min;

(3) in containing 0.5%Triton-100, incubated at room 10min cleans 3 times with PBS, each 5min;

(4) incubated at room 30min in the PBS that adds 0.5%BSA;

(5) cell is resisted under room temperature with one hatch 2h, slide is washed 3 times with PBS;

(6) add the goat anti-rabbit igg of Cy3 labelling, incubated at room 45min, PBS wash 3 times;

(7) with the mounting medium sealing that contains DAPI; Fluorescence microscope is observed down.The result sees Fig. 1～Fig. 4.Visible by Fig. 1～4: the granular cell that 10ng/mL TSA handles is compared histone H 3 K9 with H3K14 acetylation level does not have significant change with matched group, and through compare with the matched group acetylation level of histone H 3 K9 and H3K14 of the granular cell of 200ng/mL TSA processing remarkable increase is arranged all.

2.3Real time RT-PCR detects the influence of Trichostatin A to Hadc-1, Hadc-3 and P300 gene expression

2.3.1 the extraction cell total rna detects RNA concentration and purity, and according to the synthetic cDNA of M-MLV Reverse Transcriptase description record.

2.3.2Real?time?RT-PCR

(1) the mixing reagent that turns upside down uses with the slight centrifugal collection of centrifuge back then;

(2) adopt 25 μ L reaction systems, each reagent and consumption are seen table 3, and primer sequence is seen table 4:

Table 3. real-time quantitative PCR reaction system

Table 4. primer details

(3) use real time pcr amplification appearance to carry out amplified reaction, response procedures is: 94 ℃ of degeneration 3min; 94 ℃ of 10sec; 60 ℃ of 20sec; 72 ℃ of 15sec; Repeat 35 circulations, each circulates in 75 ℃ and reads plate 1s measurement fluorescence intensity; 72 ℃ of 7min; Read plate once for 65 ℃ to 94 ℃ per 0.2 ℃, draw melting curve; 72 ℃ of insulation 5min; Product and 4 ℃ of preservations.

2.3.3 data analysis

Gene quantification: as internal reference, each BIAO and BEN repeats 3 times with GAPDH (glyceraldehyde 3-phosphate dehydro-genase), and the equal Ct value of making even is pressed 2-Δ Δ Ct method analyzing gene expression.The relative concentration of mRNA is calculated through formula x=2-Δ Δ Ct; X represents the different amount of two initial substances between the group; Δ Δ Ct=Δ E-Δ C; Δ E=Ctexp-CtGAPDH exp, Δ C=Ctcontrol-CtGAPDH control, the amount of Ct value representative target gene amplification under the condition of a fixed threshold value.All data all use Means ± S.D to represent, and test at least 3 times.Expression conditions is with SPSS 16.0 softwares, Independent-Sample T Test statistical analysis.P＜0.05 is a significant difference.The result sees Fig. 5.

The result finds, handles GCs 24h with 10ng/mL TSA, and Hadc-1, Hadc-3 and p300mRNA express and compare with the blank group, and difference is remarkable (P＞0.05) not; 200ng ng/mL TSA handles GCs 24h; Hadc-1, Hadc-3mRNA express with blank group and 10ng/mL TSA processed group relatively has obvious decline; Has statistical significance; And p300mRNA expresses and with blank group and 10ng/mL TSA processed group obvious rising is arranged relatively, has statistical significance (P＜0.05).

Embodiment 3

Reagent and test kit

DMEM/F12 culture medium: Gibco BRL company; Hyclone: Lanzhou Min Hai serum company limited; Trypsin, EDTANa ₂, benzylpenicillin, streptomycin sulfate: Sigma company; The glycogen of Trizol, no RNase: Invitrogen company; M-MLV Reverse Transcriptase:Promega company; Oligo dT, dNTP MixtureRNase inhibitor, SYBR Premix Ex TaqTM: from Takara company; 2 * Taq PCR MasterMix: sky, Nanjing root Bioisystech Co., Ltd; Boric acid, agarose, Tris (Tris), bromophenol blue: the biological company limited of Nanjing great order; DEPC:SIGMA company; NaCl, KCl, NaHCO ₃, KH ₂PO4, Na ₂HPO4, DMSO, chloroform, isopropyl alcohol, dehydrated alcohol: Chemical Reagent Co., Ltd., Sinopharm Group.The cell cycle detection kit; The biological company limited of the triumphant base in Nanjing.

3.1 Flow cytometry TSA is to the influence in gonad granulocyte cycle

The granular cell that (1) will be in exponential phase is inoculated in the culture bottle, treats that cell grows to 80% and handles the pig ovary granular cell with the TSA of 0ng/mL, 10ng/mL and 200ng/mL respectively when converging, and sets up negative control group simultaneously;

(2) after the pending 24h, with 0.25% do not contain EDTA trypsin digestion cell, stop digestion with the DMEM/F12 culture medium that contains 10%FBS, it is centrifugal that (2000rpm 5min), discards culture medium;

(3) clean cell 2 times (the centrifugal 5min of 2000rpm) with PBS, collecting cell and to adjust cell concentration be 1 * 10 ⁶Individual/mL;

(4) use 70% ethanol fixed cell of 4 ℃ of pre-coolings to spend the night;

It is (5) centrifugal that (2000rpm 5min), discards fixative, cleans (the centrifugal 5min of 2000rpm) with PBS, abandons supernatant;

(6) each sample adds 100 μ L RNAse, 37 ℃ of water-bath 30min;

(7) add the dyeing of 400 μ L propidium iodides (PI), 4 ℃ of lucifuge 30min again;

(8) go up machine testing, record excitation wavelength 488nm place red fluorescence.

Table 5.TSA handles the influence to the granular cell cycle

Annotate: data are represented with means ± S.D.Significant difference between each group is with different letter representations (P＜0.05).

Utilize Flow cytometry to find, compare with the blank group, granular cell is after 10ng/mL TSA handles 24h, and each phase cell percentage ratio is compared no significant change with the blank group.And with behind the 200ng/mL TSA processing 24h; Compare with the blank group, G0/G1 phase cell percentage ratio has tangible rising (P＜0.05), and S phase cell percentage ratio significantly reduces (P＜0.05); G2/M phase cell percentage change difference is not remarkable, and the result sees Fig. 6 and table 5.

3.2Real time RT-PCR detects the influence of TSA to pig ovary granular cell Cyclin D2 and CDK4 gene expression

Method is with embodiment 2, and primer sequence is seen table 6.All data all use Means ± S.D to represent, 3 repetitions are done in each processing, and test at least 3 times.Expression conditions is with SPSS 16.0 softwares, Independent-Sample T Test statistical analysis.P＜0.05 is a significant difference.

Table 6 primer details

Use realtime RT-PCR sense cycle related gene Cyclin D2 and CDK4mRNA change of Expression, the result finds, handles 24h at 10ng/mL TSA; The expression of Cyclin D2 and CDK4mRNA and blank group compare, and difference is remarkable (P＞0.05) not, and 200ng ng/mL TSA handles 24h; What Cyclin D2 and CDK4mRNA expressed compares with the blank group; Significant change is arranged, significant difference (P＜0.05), the result sees Fig. 7.

Claims (3)

1. Trichostatin A suppresses the application in the active medicine of pig ovary granular cell in preparation.

2. application according to claim 1, the concentration that it is characterized in that Trichostatin A is 100～200ng/mL.

3. application according to claim 2, the concentration that it is characterized in that Trichostatin A is 200ng/mL.

Images